The C/EBP? transcription factor is a master regulator of myeloid development. A model of transformation to acute myeloid leukemia (AML) proposes requirement for type I mutations to stimulate proliferation and type II alterations to block differentiation. Type I changes include expression of Bcr-Abl, FLT3-ITD, or Ras activation. In isolation, these changes generate a myeloproliferative phenotype. We propose that reduction in C/EBP? expression is central to myeloid transformation, in de novo AMLs or those arising from MPD or MDS. We have developed a murine model in which a conserved 450 bp +37 kb Cebpa enhancer is flanked by loxP sites. RUNX1-ETO binds the homologous +41 kb CEBPA enhancer in human AMLs. The enhancer directs expression to GMP and to LT-HSC in a transgenic model, and Cre-mediated enhancer deletion leads to 10- fold reduction in Cebpa RNA and protein, with retention of GMP but impaired terminal myelopoiesis in vivo and indefinite myeloid colony replating in vitro. These phenotypes are reminiscent of those seen with mice expressing the truncated C/EBP?p30 variant, which develop AML by one year, though importantly our model reflects purely reduced C/EBP? activity. We hypothesize that the +37 kb Cebpa enhancer is a key mediator of normal C/EBP? expression and a key target of type II mutations in AML. We propose to evaluate the effect of enhancer deletion on normal hematopoiesis and to determine whether consequent reduction in C/EBP? leads to AML, alone or potentially more rapidly when combined with FLT3-ITD. Mice lacking C/EBP? due to biallelic open reading frame deletion do not develop AML. Residual C/EBP? retained in our murine model may be required for generation of GMP as a substrate for myeloid transformation while preventing their further maturation. Our specific aims are: AIM 1. Determine the effect of Cebpa +37 kb enhancer deletion or mutation on normal hematopoiesis. We will assess dependence of Cebpa mRNA expression on the +37 kb enhancer in stem/progenitor subsets, will assess the effect of enhancer deletion on stem/progenitor proliferation, survival, homing, and differentiation, and will assess the effect of enhancer mutations via CRISPR on Cebpa expression and enhancer epigenetics. AIM 2. Determine whether enhancer deletion facilitates de novo transformation or MPD progression. We will determine whether enhancer deletion alone, mediated by Mx1-Cre or Vav-Cre, allows development of AML, will determine whether Cebpa enhancer deletion leads to AML when combined with FLT3ITD, and will use RNA-seq to identify pathways affected by enhancer deletion, alone or with FLT3ITD, in preleukemic stem/progenitors. AIM 3. Determine whether the Cebpa enhancer is targeted epigenetically in murine/human MDS/AML. We will assess pre-leukemic and leukemic enhancer activity and epigenetics in marrow expressing RUNX1-ETO9a, will conduct a similar analysis during myeloid transformation following MDS induced by NUP98-HOXD13, and will determine whether the CEBPA enhancer has reduced activating and increased repressive epigenetics in human AMLs, correlated with CEBPA mRNA expression.